ACYLATION TARGETS ENDOTHELIAL NITRIC-OXIDE SYNTHASE TO PLASMALEMMAL CAVEOLAE

Endothelial nitric-oxide synthase (eNOS) generates the key signaling m olecule nitric oxide in response to intralumenal hormonal and mechanic al stimuli. We designed studies to determine whether eNOS is localized to plasmalemmal microdomains implicated in signal transduction called caveolae. Using immunoblot analysis, eNOS protein was detected in cav eolar membrane fractions isolated from endothelial cell plasma membran es by a newly developed detergent-free method; eNOS protein was not fo und in noncaveolar plasma membrane. Similarly, NOS enzymatic activity was 9.4-fold enriched in caveolar membrane versus whole plasma membran e, whereas it was undetectable in noncaveolar plasma membrane, 51-86% of total NOS activity in postnuclear supernatant was recovered in plas ma membrane, and 57-100% of activity in plasma membrane was recovered in caveolae. Immunoelectron microscopy showed that eNOS heavily decora ted endothelial caveolae, whereas coated pits and smooth plasma membra ne were devoid of gold particles. Furthermore, eNOS was targeted to ca veolae in COS-7 cells transfected with wild-type eNOS cDNA. Studies wi th eNOS mutants revealed that both myristoylation and palmitoylation a re required to target the enzyme to caveolae and that each acylation p rocess enhances targeting by 10-fold. Thus, acylation targets eNOS to plasmalemmal caveolae, Localization to this microdomain is likely to o ptimize eNOS activation and the extracellular release of nitric oxide.